Decoding efficacy and resistance space at a drug binding site.

Assessing and understanding the impacts of all possible mutations at a drug binding site remain challenging. Here we use multiplex oligo targeting for mutational profiling, and computational modelling, to decode efficacy and resistance space at the otherwise native binding site for an anti-trypanosomal proteasome inhibitor. We saturation-edit twenty codons in the Trypanosoma brucei proteasome and subject the resulting libraries to stepwise drug selection and codon variant scoring, yielding dose-response profiles for >100 resistance-conferring mutants. Codon variant scores are predictive of relative resistance observed using a bespoke set of mutants, while fitness profiling reveals otherwise extensive constraints on mutational fitness and resistance space. The resistance profile is predictive of routes to spontaneous drug resistance observed within 'accessible', single nucleotide mutational space, while in silico predictions are closely aligned with impacts on drug resistance observed in cellulo. Thus, multiplex oligo targeting facilitates assessment of all possible mutations at a drug binding site.